Utilisation of NMR to study N-myc TAD dynamics and interactions

The myc family of proteins (c-, N- and L-myc) are transcription factors (TFs) responsible for maintaining the proliferative program in cells. 28% of tumours exploit these properties through deregulation of myc in various ways, which aids cancer through faster growth rates. Structural and biochemical knowledge of c-myc is lacking, with even less studies performed on N-myc, which is one of the main drivers of paediatric tumours. N-myc tumourigenic functions are mediated via intrinsically disordered transactivation domain (TAD), which makes N-myc TAD a suitable candidate for nuclear magnetic resonance (NMR) studies. NMR collects in-solution data on per-residue basis and can handle a dynamic ensemble of structures. Firstly, N-myc TAD backbone assignment was obtained, with the spectral resonant frequencies assigned to individual residues within primary amino acid sequence of N-myc TAD. Chemical shift index analysis, computer modelling and relaxation data revealed that N-myc TAD is mostly intrinsically disordered with elements of transient secondary structures. Myc is rapidly degraded following mitogen signalling, through a hierarchy of post-translational modifications (PTMs). This work recreated these PTMs in vitro using NMR and confirmed that N-myc is subjected to the same degradation mechanism as c-myc. By employing various biochemical methods, putative partners of N-myc TAD were interrogated, which revealed that N-myc and c-myc do not have the same interactome. The novel data presented in this work underline the necessity for tailored research towards N-myc, as N-myc and c-myc might differ in their modality of interactions with binding partners, despite a degree of conservation between the two proteins. This fundamental research will aid our understanding of the mechanism by which the TAD and other intrinsically disordered protein regions utilise transient/latent structures to bind a wide range of partners, despite lacking a stable 3D structure.